Terpenes constitute a large group of structurally diverse molecules synthesized naturally by organisms as diverse as bacteria, fungi, plants and animals. Much research has been concentrated on the biochemistry and biological functions of terpenes or their derivatives for potential commercial exploitation (Gershenzon and Dudareva 2007 Nat Chem Biol 3:408).
The result of these studies is a variety of terpene-based products ranging from pharmaceuticals, such as anti-cancer drug paclitaxel and anti-malaria drug artemisin, to fragrances and aroma ingredients, such as menthol and patchuol.
In the field of plant breeding, there is an interest in terpene secondary metabolites produced by many plant species to resist pathogens, to repel or kill pests or to attract beneficial organisms, e.g., predators or parasitoids of pest insects or plant pollinators, or other organisms. Wild plant species frequently produce beneficial secondary metabolites lacking in their cultivated relatives, and therefore are an important source of traits for introgression into cultivated varieties. It is known that secondary metabolites, such as volatile terpenoid compounds, can directly influence insect behavior (Bruce et al., 2005 Trends Plant Sci 10:269-274). For instance, methyl ketones and sesquiterpene carboxylic acids identified in Solanum habrochaites and acyl-glucose esters from Solanum pennellii were found to be toxic to different insect classes, such as Lepidoptera, mites, and aphids (Williams et al., 1980 Science 20:888; Goffreda et al., 1990 Plant Cell 2:643; Juvik et al., 1994 J Econ Entomol 87:482; Frelichowski and Juvik, 2001 J Econ Entomol 94:1249). Often mono- and sesquiterpene hydrocarbons, sesquiterpene acids, methylketones and sugar esters are accumulated in plants in specialized organs such as glandular trichomes on stems and leaves. Several studies correlated the density of glandular trichomes with levels of resistance to pest insects, e.g., maize earworm Heliothis zea and Colorado potato beetle (Kauffman and Kennedy, 1989 J Chem Ecol 15:1919-1930; Antonius et al., 2001 J Environ Sci Health B 36:835-848; Antonius et al., 2005 J Environ Sci Health B 40:619-631). The methylketones 2-undecanone and 2-tridecanone accumulated in glandular trichomes of S. habrochaites were shown to be toxic to larvae of Colorado potato beetle and adult whiteflies B. tabaci, respectively (Antonius et al., 2005 J Environ Sci Health B 40:619-631). The myrtle oil, including the monoterpene linalool among its essential components, was shown to have an insecticidal effect on bean weevils, Acanthoscelides obtectus Say (Coleoptera: Bruchidae) (Ayvaz et al., 2010 J Insect Sci 10: 1536-2442). The sesquiterpenes zingiberene and curcumene, and the monoterpenes p-cymene, α-terpinene, and α-phellandrene from wild tomato S. habrochaites and S. pennellii, respectively, were shown to have insecticidal properties (Bleeker et al., 2009 Plant Physiol. 151:925). Bio-assays have demonstrated that the sesquiterpenes 7-epizingiberene and its derivative R-curcumene repelled adult whiteflies from landing on tomato plants (Bleeker et al., 2011 Phytochemistry 72:68), and that plants with endogenous production of zingiberene showed resistance to Tuta absoluta. (De Azavedo et al., 2003 Euphitica 134:247-251).
Genetic inheritance of the genes associated with development of different types of glandular trichomes and production of zingiberene was studied in interspecific crosses between S. lycopersicum, a cultivated tomato which does not produce zingiberene, and S. habrochaites, a wild species with high zingiberene production. In F2 plants from these crosses, zingiberene content correlated with resistance to B. tabaci. This study suggested feasibility of breeding plants with high levels of zingiberene, 2-tridecanone, and/or acylsugars, which would lead to high levels of resistance to whiteflies (Freitas et al., 2002 Euphytica 127: 275-287). However, programs on introgression of useful traits into cultivated varieties are time consuming and costly, therefore production of secondary metabolites in plants lacking them or elevating levels of these metabolites in plants synthesizing them—albeit in insufficient levels—is an attractive goal.
The biosynthesis of terpenes in plants has been extensively studied and many genes coding for the pathways steps from precursors to final products were discovered (Wither and Keeling, 2007 Agro Microbial Biotechnology 73:980-990; Sallaud et al., 2009 Plant Cell 31:301).
Due to the widespread infestation of crop and ornamental plant species with pest insects such as B. tabaci and the greenhouse whitefly Trialeurodes vaporarium, resulting in great economic losses, means of regulating plant natural defense molecules to repel pests has received a renewed interest of scientists and plant breeders.
It is known that manipulation of transcription factors can regulate complex pathways in animals and plants involving numerous target genes. This may result in increased expression of useful compounds. Alternatively, blocking transcription factors may lead to decreased or completely suppressed production of undesirable compounds and/or removal of unwanted traits.
Several transcription factors controlling genes involved in plant secondary metabolism were identified, cloned and showed high efficiency in regulating complex metabolic pathways. For instance, the transcription factor WRKY was shown to regulate δ-Cadenine Synthase A, a sesquiterpene synthase that catalyzes the first step of pathway leading to production of gossypol in cotton (Xu et al., 2004 Plant Physiol 135:507-515).
Moreover, while overexpression of individual genes of the biosynthetic pathways was shown to provide limited success, perhaps, due to poor substrate availability, genome-wide expression of the flavonol-specific transcription factor, AtMYB12, in tobacco not only regulated the phenylpropanoid pathway, but also modulated other metabolic pathways that led to increased flux availability to this pathway, and eventually to an increased resistance against Spodopter lituralis and Helicoverpa armigera insects (Misra et al., 2010 Plant Physiol 152: 2258-2268).
MYB transcription factors have been indicated also to activate multiple enzymes required for production of glucosinolates, crucifer-specific secondary metabolites, in Arabidopsis. MYB51 was shown to activate the indolic glucosinolates biosynthesis and confer enhanced resistance to the herbivorous pest Spodoptera exigua in plants overexpressing it (Gigolashvili et al., 2007 Plant J 50: 886-901). Other MYB transcription factors, such as MYB76, MYB28 and MYB29, are shown to regulate enzymes involved in the production of aliphatic glucosinolates (Gigolashvili et al., 2007 Plant J 51: 247-261; Gigolashvili et al., 2008 New Phytol 177:627-642).
There is a need in the art to provide transcription factors regulating terpene biosynthesis, in particular transcription factors that have an effect on the biosynthesis of mono- or sesquiterpenes in plants or other organisms leading to the production of terpene compounds that repel or attract insects, or other organisms.